In this study, a technique for particle streamwise timing, spacing and velocity control (alignment) in microchannel flow by controlling the forces exerted on the particle in space and time, was developed. In the present technique, the timing of particles crossing a certain position in microchannel flow with a specific interval and the particle velocity are controlled by applying acceleration and deceleration forces periodically in the streamwise direction and activating them periodically. The force is produced by a dielectrophoretic force using ladder-type electrodes embedded in the microfluidic device and is turned on and off in a cycle. The timing of particles crossing a certain position can be changed by adjusting the phase of the on-off cycle, i.e., the phase of the voltage signal. In the experiment, timing and velocity were measured at the inlet and outlet of ladder-type regions for Jurkat cells and particles of some variation in size, and probability density functions for the deviation of these values from the equilibrium (aligned) state were evaluated. Further, we will discuss the motion characteristics of the particles numerically and experimentally to understand the mechanism and evaluate the performance of the particle timing control and alignment using the present technique. The results confirm that the particles randomly distributed at the inlet of ladder-type electrode regions are controlled to flow with even spacing at a specific velocity. Moreover, the timing of the particles passing a specific location in the ladder-type electrode region was synchronized with the activated/nonactivated cycle of the applied force and could be specified.